Alain Locatelli

Melatonin Enters the Cerebrospinal Fluid through the Pineal Recess

The pineal recess (PR), a third ventricle (IIIV) evagination penetrating into the pineal gland, could constitute a site of melatonin passage to the cerebrospinal fluid (CSF) and explain the high concentrations of melatonin in this fluid. To test this hypothesis, we characterized melatonin distribution in the IIIV of sheep by CSF collection in the ventral part of IIIV (vIIIV) and in PR. At 30 l/min collection rate, melatonin concentrations were much higher in PR than in vIIIV (19,934 6,388 vs. 178 70 pg/ml, mean SEM, respectively, P < 0.005), and they increased in vIIIV when CSF collection stopped in the PR (P < 0.05). At 6 l/min, levels increased to 1,682 585 pg/ml in vIIIV and were not influenced by CSF collection in the PR. This concentration difference between sites and the influence of PR collection on vIIIV levels suggest that melatonin reaches the PR and then diffuses to the IIIV. To confirm the role of PR, we demonstrated that its surgical sealing off decreased IIIV melatonin levels (1,020 305 pg/ml, compared with 5,984 1,706 and 6,917 1,601 pg/ml in shams or animals with a failed sealing off, respectively, P < 0.01) without changes in blood levels. Therefore, this study identified the localization of the main site of penetration of melatonin into the CSF, the pineal recess. (Endocrinology 143: 84 –90, 2002)

Production, freezing and transfer of embryos from a bluetongue-infected goat herd without bluetongue transmission

In order to import non-seasonal Creole goats from the Carribean to Europe for an experimental purpose, thirty Creole goats were treated with 10 mg of FSH; embryos were collected at slaughter, washed and deep frozen. After rapid thawing, they were reimplanted surgically into European dairy goats. Twenty-four females ovulated but only 17 of the ovulating females had functional corpora lutea (CL) at collection. Ovulation rate (CL goat ) and recovery rate (embryo CL ) were 13.8 and 78% for females with functional CL. Of 191 embryonic structures collected, 79% were considered suitable for deep freezing: 23% were young blastocysts, 47% were expanded blastocysts, and 30% were zona-pellucida (zp)-free and zp-damaged embryos. Seventy-eight embryos were thawed and 63 were reimplanted. Sixty-eight percent of the recipient females delivered 19 kids. The percentage of kids born relative to good-quality re-implanted embryos was higher for zp-free embryos (64%) than for young and expanded blastocyts (36%). Forty-seven percent of the donor females had strong positive serological reactions for bluetongue virus antibodies against serotypes 6 and 14. However, no recipient goats or newborn kids were positive. Virus isolation attempts on the collection media and last embryo washes were negative.

Effect of long-term cocaine administration to pregnant ewes on fetal hemodynamics, oxygenation, and growth.

Objective To assess uterine and fetal blood flows by Doppler velocimetry and fetal growth and oxygenation in pregnant ewes treated daily with cocaine and to determine whether cocaine impairs fetal cardiac and cerebral reactivity. Methods The study groups received 70 mg (n = 7) or 140 mg (n = 7) of cocaine and the control group (n = 7) received placebo injected intramuscularly daily on days 60–134. Hemodynamic data were measured at rest and during two acute hypoxic tests at cesarean delivery performed on day 134. Results The fetal heart rate (FHR) and umbilical and uterine resistance indices (RIs) were higher in the cocaine groups than in the control group (FHR: 187 ± 8 and 166 ± 8 beats per minute at 83 and 123 days, respectively, in controls and 9–11% higher in cocaine groups; umbilical RI: 0.79 ± 0.06, 0.60 ± 0.04, and 0.52 ± 0.06, at 83, 105, and 123 days, respectively, in controls and 11–17% higher in the cocaine groups [P <.01]; and uterine RI: 0.40 ± 0.05, 0.40 ± 0.04, and 0.37 ± 0.04, at 83, 105, and 123 days, respectively, in controls and 13–35% higher in cocaine groups [P <.05]). At delivery on day 134, the following characteristics were found to be different in the cocaine groups: fetal weight (4.03 ± 0.2 kg in controls and 15–21% lower in the cocaine groups [P <.02]), partial pressure of oxygen (26.5 ± 1.4 mmHg in controls and 15–16% lower in cocaine groups [P <.05]), umbilical RI (0.40 ± 0.03 in controls and 11–17% higher in cocaine groups [P <.01]), cerebral RI (0.61 ± 0.03 in controls and 9–15% lower in cocaine groups [P <.01]), and cerebral-umbilical ratio (1.52 ± 0.04 in controls and 22–23% lower in cocaine groups [P <.001]). During the hypoxic tests, the cerebral RI (P <.05) and the cerebral-umbilical ratio (P <.05) decreased significantly less in the two cocaine groups. The FHR response was reduced significantly less in the two cocaine groups (P <.05). Conclusion Long-term exposure to cocaine induces uterine and fetal blood flow disorders, fetal growth restriction, and hypoxia. It reduces the capability of the cerebral vessels to vasodilate and the heart rate to increase during acute hypoxia.

Brain damage and hypoxia in an ovine fetal chronic cocaine model.

OBJECTIVE To assess the development of brain damage in an ovine fetal chronic cocaine model. To evaluate the effect of isolated hypoxic tests on this model and to correlate hemodynamic findings (brain-sparing effect) following fetal hypoxia and the occurrence of brain damage. STUDY DESIGN Fifteen ewes were divided into a control group (n=7) and a cocaine treated group (n=8). From day 65 to day 134 the cocaine treated animals received a daily (5 days per week) intramuscular injection (2 mg/kg cocaine) and the control animals a placebo injection (2 ml of isotonic solution). Both groups underwent hypoxic tests (cord compression (3 min) and aortic compression (1 min)) at 90 and 134 days. In addition, anesthesia for magnetic resonance imaging (MRI) examination was carried out at 125 days. Fetal blood samples were collected during both series of hypoxic tests and the cerebral and umbilical flows were monitored by Doppler. Samples from 25 brains (control n = 10; cocaine n= 15) were processed for light and electron microscopic examination. Quantification of brain damage was done on semithin sections from six areas of cortex and germinal matrix on each fetus. RESULTS Similar forms of brain damage (selective neuronal loss limited to the parasaggital cortex, striatum, hippocampus and Purkinje cells) was present in both groups but lesions were more frequent in the cocaine treated group as shown by quantitative analysis for the proportion of abnormal capillaries (65% vs. 35%), capillary edema (61% vs. 34%) and abnormal neurons showing delayed neuronal degeneration (DND) (66% vs. 36%) in the cocaine and control group respectively. There was no significant difference in immunoreactivity for glial fibrillary acidic protein (GFAP) but it was more marked in the cerebellum of cocaine treated animals. Fetal blood samples showed a moderate sustained hypoxia and Doppler findings demonstrated the presence of a brain sparing effect associated with increased uterine and umbilical vascular resistance in the cocaine treated group. Nevertheless, the amplitude of the heart rate increase and cerebral dilatation was significantly lower in the cocaine treated animals. CONCLUSION This ovine fetal chronic cocaine model showed the presence of brain damage. Cocaine treatment seems to potentiate the effect of the hypoxic tests. Independent of the cause, the brain damage developed in the presence of brain sparing effect, strongly suggesting that this phenomenon is a sign of a pathological fetal condition and no guarantee that it will prevent tissue damage.

Cerebral MRI on fetuses submitted to repeated cocaine administration during the gestation: an ovine model.

The aim of this study was to determine the role of Magnetic Resonance Imaging (MRI) in investigating fetal cerebral lesions induced by long term exposure to cocaine during sheep pregnancy. Cerebral Magnetic Resonance Imaging was performed on two groups of fetuses at 125 days of gestation (normal gestation: 145 days). The control group consisted of eight fetuses of four pregnant ewes. The study group consisted of eight fetuses of four pregnant ewes receiving daily 140 mg/kg injection of cocaine from day 60 until delivery. The following MR sequences were applied: T1-weighted FLASH, and T2-weighted Fast-Spin-Echo. Cerebral images were evaluated semi quantitatively using the following criteria: Heterogenicity, contrast between grey and white matter, contours irregularity, hyposignal, lateral ventricle sizes. The brightness distribution and homogenicity of the images were analysed by means of edge pair distributions using a new computerized method originally designed for ultrasound images analysis developed by Ultrasight inc (USA). (1) Flash T1: Heterogenic areas and irregular contours were more frequent in cocaine exposed fetuses. The contrast between grey and white matter was more important in the cocaine group. Hyposignal was found only in the cocaine group. Enlarged lateral ventricle occurred more frequently in the cocaine group. (2) Spin echo T2: The contrast between grey and white matter was higher and the contours of the brain more irregular in the cocaine group. Heterogenicity and hyposignal were also more frequent in this group but the difference with the control group was not significant. The computerized analysis of the contrast density on the cerebral images showed that 88% of the areas exceeding the reference level concerned the cocaine group, while only 14% of the areas exceeding the reference level concerned the control group. Long term exposure to cocaine induces cerebral tissue modifications, in favor of an advanced maturation and the development of hypoxic lesions. The histology of the brains confirmed in the cocaine group, the existence of hypoxic lesions with gliosis, perivascular edema and hemorrhages, and neuronal death.

Corpus luteum derived copper, zinc-superoxide dismutase serves as a luteinizing hormone-release inhibiting factor in sheep.

In the present study, we report the purification and characterization of a polypeptide from the sheep corpus luteum of pregnancy with a potent luteinizing hormone-release inhibiting factor (LH-RIF) bioactivity that stained as a single band in SDS-PAGE with an apparent molecular mass of 16000 Da. The amino acid sequences obtained after sequence analysis of peptides derived from the trypsin digestion of LH-RIF were subjected to a protein data bank search and were found to be identical with regions of sheep copper, zinc-superoxide dismutase (Cu,Zn-SOD). The measured mass of LH-RIF (15604.2+/-1.9 Da) was found to be similar to the theoretical mass of sheep Cu,Zn-SOD (15603.5 Da), with a disulfide bond and N acetylated alanine at the N-terminus. The inhibitory action of Cu,Zn-SOD on pulsatile LH secretion would suggest that this antioxidant may play an important role, either independently or in concert with some neurotransmitters, in the neuroendocrine regulation of sheep female reproductive function.

Pineal Microdialysis of the Melatonin in Conscious Sheep: Methodology, Application to a Diurnal Rhythm and Effect of Isoproterenol

This paper describes the development of a new technique to measure melatonin contents in the pineal gland of moving sheep: the microdialysis. A dialysis probe was used to collect extracellular fluid in the sheep pineal gland, but also to inject directly into it different drugs such as isoproterenol at a very low concentration. The probe was implanted the day before the beginning of the experiment in order to obtain low levels of melatonin. This technique makes it possible to measure melatonin in the dialysate and plasma of rams submitted to 8L:16D. No melatonin either in the dialysate or in the plasma was found during the light phase. Shortly after lighting off, the melatonin concentration increased in the dialysate and plasma and remained stable during the dark phase. Melatonin concentrations began to decrease before lighting on and no detectable levels were found during the following light phase. The secretion of melatonin is, at least, under adrenergic regulation. Local infusion of isoproterenol (90 μl at 10−6 M), an agonist of β adrenergic receptor, through the probe, increased melatonin levels during 2 h, even when infusions were repeated 3 times. This demonstrates the presence of β adrenergic receptors. The technique presented in this paper could be of considerable interest for studying sheep pineal gland and its main secretion, melatonin, for example during diurnal rhythms or for studying its regulation.

Intracerebroventricular administration of copper-zinc superoxide dismutase inhibits pulsatile luteinizing hormone secretion in ovariectomized ewes

Intracerebroventricular (i.c.v.) injection of an inhibitor of nitric oxide synthase (NOS) abolishes pulsatile luteinizing hormone (LH) secretion. It has been demonstrated that structural and functional analogs of copper-zinc superoxide dismutase (Cu,Zn-SOD) inhibit neuronal NOS. The present study examined the ability of Cu,Zn-SOD to affect pulsatile LH release in the ewe. Bovine Cu,Zn-SOD was administrated into the third cerebral ventricle of unanesthetized, freely moving, ovariectomized (OVX) ewes. Jugular blood samples were taken every 15 min for 5 h before and 8 h after i.c.v. injections. In a pilot trial using three OVX ewes, i.c.v. injection of Cu,Zn-SOD at a dose of 0.5, 1.0 or 2.0 microg in 100 microl saline decreased plasma LH levels and abolished LH pulses, without affecting FSH secretion. In the main experiment, i.c.v. injection of 100 microl saline had no effect on mean LH levels and LH pulse frequency, whereas i.c.v. injection of Cu,Zn-SOD at a dose of 1 microg/100 microl saline significantly (P < 0.01) decreased mean LH levels and LH pulse frequency. In conclusion, this study provides the first evidence for the role of Cn,Zn-SOD in the control of LH secretion at the level of the brain in female mammals.

A study of ovarian activity in the ewe using chronic catheterization of the utero-ovarian vein

Eighty-six adult, cyclic, anestrous and postpartum Ile-de-France ewes were placed under general anaesthesia. A catheter was inserted in the utero-ovarian vein (u.o.v.) of one or both ovaries in each ewe. The animals were kept unrestrained in pens and allowed to experience normal maternal behaviour, including suckling. Oestrus was detected with rams. Out of 167 catheters inserted, 70.7% allowed repetitive blood collection, 24.0% were obstructed for short periods and 5.4% remained completly blocked. Oestradiol- 17beta was assayed without extraction from the u.o.v. plasma samples. From these results, we conclude that this technique is suitable for investigating the mechanisms involved in the recovery of cyclical ovarian activity after parturition, particularly because the follicular response to endogenous gonadotropins can be assessed. This is not possible when plasma samples are obtained from a jugular vein.